Homeostasis of cellular proteins is maintained through a combination of synthesis and degradation. The pathway that accounts for the majority of protein degradation is the ubiquitin-proteasomal pathway. Ubiquitin (Ub) is highly conserved in all cells and the generation of a multi-Ub chain typically targets proteins for degradation by the proteasome. However, ubiquitination is highly reversible more ..

Homeostasis of cellular proteins is maintained through a combination of synthesis and degradation. The pathway that accounts for the majority of protein degradation is the ubiquitin-proteasomal pathway. Ubiquitin (Ub) is highly conserved in all cells and the generation of a multi-Ub chain typically targets proteins for degradation by the proteasome. However, ubiquitination is highly reversible and dynamic. Deubiquitination, the reverse process, is catalyzed through the action of enzymes referred to as isopeptidases or deubiquitinating enzymes (DUBs) [1, 2]. This group of enzymes is collectively responsible for maintaining adequate pools of free ubiquitin and regulating the ubiquitination status of cellular proteins. The class of DUBs referred to as the ubiquitin-specific proteases (USP) family functions endoproteolytically to cleave Ub chains from a wide range of protein substrates. USP2a deubiquitinates fatty acid synthase (FASN) which has recently been identified as an emerging oncology target [3-6]. To identify inhibitors of USP2a a cell-free assay was employed.

This assay takes advantage of attaching an Ub or UBL (ubiquitin-like protein) to a reporter enzyme, rendering the reporter catalytically inactive. The reporter enzyme chosen is phospholipase A2 (PLA2), which has an absolute requirement for a free amino terminus. Thus, fusion of a UBL to the N-terminus of PLA2 inactivates PLA2. When the UBL-PLA2 reporter enzyme is cleaved by USP2, the activated reporter can subsequently act on its substrate, which is the commercially available NBD C6-HPC (Invitrogen). Thus, this coupled assay gives a fluorescent read-out, which is generated through the cleavage of the PLA2 reporter enzyme. The assay, developed by Progenra, Inc. uses Ub-PLA2 and USP2a at near equal and low (nM) amounts and has been licensed for sale by LifeSensors, Inc. as the Ub-IsoPro1 kit.

The assay buffer, prepared fresh at the day of the assay, contains 20mM Tris-HCl pH 8.0, 2mM CaCl2, 2mM beta-Mercaptoethanol, 0.1% DMSO. 3ul of 40nM (20nM final) USP2 core, which is stored on ice, is dispensed into a medium-binding black solid Kalypsys 1,536 well plate using the Kalypsys dispenser. The assay plate is then pinned with 23nL compound with the Kalypsys pintool in columns 5-48. The controls are pinned as follows: column 1&2 two-fold dilutions of 2M NEM in duplicate; column 3 DMSO; column 4 2M NEM (final concentration 38.2mM). The assay plate is incubated at RT for 30min. Subsequently, 3uL of 40nM (20nM final) Ub-PLA2 and 40uM (20uM final) NBD C6-HPC (stored on ice and protected from light) were dispensed into the plate using the Kalypsys dispenser. Plates were read immediately (0 Hour) on an Envision (Perkin Elmer) plate reader using the following wavelengths Ex 460nm /Em 535nm. Then, the assay plates were incubated for 2.5 hours and read again on the Envision using the same settings

Data were normalized to the to AC100 inhibition (NEM). Concentration-response curves were fitted to the normalized data and the concentration-response curves were then classified based on curve quality (r2), response magnitude and degree of measured activity. The time zero reading was used to flag fluorescence artifacts.

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.

Float

μM

3

Efficacy

Maximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.

Float

%

4

Analysis Comment

Annotation/notes on a particular compound's data or its analysis.

String

5

Curve_Description

A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically signficant, but below 80% of control.

String

6

Fit_LogAC50

The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).

Efficacy at zero concentration of compound from a fit of the data to the Hill equation.

Float

%

11

Fit_CurveClass

Numerical encoding of curve description for the fitted Hill equation.

Float

12

Excluded_Points

Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.